Anaerobic bacteria of the genus Clostridium secrete seven botulinum neurotoxin (BoNT) serotypes (A-G), which enter human neuronal cells and destroy proteins essential for synaptic vesicle fusion to the presynaptic membrane. Neurospecificity and presence of proteolytic subunits underlie the fact that these toxins pose a significant health and bioterror threat. Although BoNTs can be neutralized extracellularly with anti-toxin preparations, the proteolytic subunits escape current treatments once inside human motoneurons. To find intracellular protease inhibitors, cell-based assays suitable for high-throughput drug screening are urgently needed. To this end, we have developed yeast strains that are susceptible to the lethal effects of two BoNT proteases, serotypes B and C. We have expressed these proteases under control of a promoter that can be regulated in yeast, resulting in cell growth when the promoter is repressed and cell death when the promoter is derepressed. The current application is focused on modifying our yeast strains genetically to achieve the sensitivity required for high-throughput drug screening. Specifically, we will (i) modulate levels of the BoNT proteases inside cells while maintaining regulated control, (ii) increase yeast-membrane permeability to small compounds, and (iii) eliminate drug-efflux pathways. The constructed yeast strains will then be subjected to high-throughput BoNT inhibitor screening by employing a library collection with over 200,000 compounds. Through this study, we will generate the first high-throughput assays for intracellular inhibitors against the highly lethal BoNT proteases and use the assays to identify urgently needed therapeutic compounds that will help eliminate the threat of BoNTs as weapons of bioterror and agents of human disease.